Diagnostic composition and method



Patented May 19 1942 DIAGNOSTIC COMPOSITION METHOD Jonas Kamlet, Brooklyn, N. Y., assignor to Miles Laboratories, Inc., Elkhart, Ind, a corporation of Indiana N Drawing. fApplication October 2, 1940, Serial No. 359,345

7 6 Claims.

The present invention relates to diagnostic compositions and, more particularly, to diagnostic compositions suitable for use in thequalitative detection and quantitative estimation of acetone and acetoacetic acid in body fluids (e. g.

urine, blood, cerebrospinal fluid). .Ithasfor its object ot provide a simple, rapid and convenient method for performing-these tests with a high degree of accuracy and specificity, and one that can readily beused by the average physician without laboratory equipment or specialized analytical training.

The detection and estimation of acetone and acetoacetic acid are of the greatest importance in diagnosing a number of pathological conditions,

such as diabetes, mellitus, malnutrition, cachexia, various gastrointestinal disorders, high fevers of long duration, etc. However, the methods here tofore employed require not only some technical ability and experience, but also make use of equipment and chemicals which are not always available to the average medical practitioner. Thus, the qualitative methods described by Lange, Le Noble, Rothera,'Rantzman and Kamlet based on the reaction of sodium nitroprusside with ammonia or monoethanolamine all require the use of freshly prepared reagents anda good analytical technic. The various quantitative methods (Van Slyke and Fitz, Marriott, Behre and Benedict) all require skill and equipmentrarely found outside of a hospital laboratory. Fortune, United States Patent No. 2,186,902, has described a urine acetone test based on the reaction of diluted urine with a soluble nitroprusside, an ammonium salt, a soluble carbonate and a material (such as tale) that will cause the observed color to be that due to reflected light. Thismethod is not,"however, suitable for-the detection of acetone and acetoacetic acid in deproteinized blood filtrate or in cerebrospinal fluid, where it can be of value in the diagnosis of meningitis.

The present invention has for its further object to provide a means whereby an unskilled person may perform a quantitative or qualitative estimation of acetone and acetoacetic acid in urine, cerebrospinal fluid or deproteinized blood filtrate,

without the use of any equipment except a test tube. It will enable the average physician to follow the course of his patients disease and to judge the efiectiveness of his treatment. The appearance of a ketosis, the phenomenon of excessive acetone and acetoacetic acid production by the body, is always a danger signal that warns of an abnormal state of metabolism that is apt to seriously endanger the healthlofthe patient ifnot properly and promptly diagnosed and properly treated. A simple acetone test that, can be performed by the patient proper is therefore of the greatest value in preventingsuch conditions.

.This is especially true in those diabetics that require insulin injections two or three times during the day. I I

The most accurate method heretofore available forthe qualitative detection and quantitative estimation of acetone, and acetoacetic acid is that of'Behre and Benedict (Journ. Lab. Clin. Med., 13, 770, 1928; Journ. Biol. Chem.,. 70, 187, 1926).

This method is based on the reaction of acetone with salicylaldehyde in alkaline solution to give '''the .deeplycolored, orange to red compound,

salicylalacetone. .Any acetoacetic acid in the solution is converted quantitatively to acetone by the alkali. v

This method has never, however, been suitable for use by the average physician or unskilled patient. salicylaldehyde is an unstable liquid that H is highly irritating to the mucous membranes (Wohler & Frerichs, Annalen der Chemie, vol; 65, page 336-), .while the concentrated sodium hydroxide solutions required rapidly become attenuated by' the absorption of carbon dioxide metaland alkali-earth-metal-bisulfite addition products of salicylaldehyde and}! member of the group consisting of the oxides and the hydroxides of the alkali-metals and the alkali-earth metals, 'both in the dry state. On reaction with an aqueous fluid, such as urine, cerebrospinal ,fluid or blood, the bisulfite addition product of salicylal:

dehyde will react with the alkali, liberating free salicylaldehyde. In the presence of-free alkali, the latter will then interact with any acetone present in the fluid togive the characteristic coloration. p p The alkali metaland alkali-earth metal bisulfite addition products of salicylaldehyde are obtained by dissolvin equimolecular proportions of the bisulfite and salicylaldehyde in hot water, and allowing .to crystallize. The addition products form stable, white compounds, very soluble in water, but slightly solubleor insoluble in all organic solvents. When reacted with water in the acetoacetic acid), the color of the urine will change to orange or red. In the absence or acetone bodies, the urine will retain the characteristic yellow color of the sodium salt of salicylaldehyde.

To determine quantitatively the amount of acetone in the fluid, the orange or red color ob- I served (which is strictly proportional in intensity to the .amount of total acetone and 'acetoacetic acidoriginally present) is comparedwith a color chart of known concentrations 'of acetone in urine. This method of comparison is a direct corollary of Beers law, on which the entire art of colorimetry. is based; namely-that the amount of light absorbed in passingthrough a colored medium. is directly proportional to the concentra:- tion of colored substance. The results of this quantitative colorimetric testv may be stated in terms of milligrams per 100 cc. or as a percentage or ratio as desired.

It is obvious, of course, that diluents, excipients, dispersing agents and auxiliary substances may be incorporated into these compositions without changing the basis of the present invention. Diluents such as talc and kaolin or an alkaline metal silicate may be added to obtain'a better tablet or a more stable product. Excipients such as lactose, starch and flour may be added'to f acilitate the formation of granules. Dispersing agents such as saponin,gelatin, agar, soap, tragacanth and gum arabic may be added to accelerate the disintegration of the tablet when placed into the fluid. Auxiliary substances, such as coloring agents or perfumes may likewise be added." These compositions may be used in the form of powder, or granules, or dispensed in capsules, or they can be pressed into tablets of such 'a size as is convenient for the performance of a single test.

The following example of atypical composition covered bythis invention is intended to define and illustrate, but in no way to limit this invention. to the reagents, proportions or conditions described therein. Obvious modifications will occur to any person skilled in the art.

7 7 Example v y To a solution of 1050 grams of sodium bisulfite in 5 liters of boiling water is added 1250 grams of technical salicylaldehyde.

The mixture is stirred vigorously for one hour and then steamdistilled-until a test-portion of the distillate fails to show the presence of a phenol. The hot solu-- tion is then evaporated in vacuo to a volume of two liters and allowed to cool. The copious precipitate of salicylaldehyde sodium bisulfite that forms on standing overnight, is filtered .oif,

' washed with alcohol and ether and dried.

mixture containing essentially the sodium bi- When the mixture has become homogeneous, it is pressed into five-grain tablets.

To facilitate the disintegration of these'tablets,

a mechanical dispersing agent may be incorporated. This dispersing action may also be accomplished by incorporating into the composition a dry compound which, when added to water,

will react vigorously with the caustic soda and effect the mechanical disintegration of the tablet,

Examples of such compounds are citric acid, tartaric acid, oxalic acid, potassium bisulfate,monobasic calcium phosphate, etc. In each case, it is necessaryto have a large excess of caustic soda over that amount required for the stoichiometric neutralization of the acidic substance.

The sensitivity of this composition is such that it will detect 0.050% of acetone in urine, or 0.035% of acetone in aqueous solution.

Having described my invention, what I claim and desire to protect by Letters Patent is:

1, Diagnostic compositions comprising a drycontent of acetone 'and'acetoacetic acid.

2. Diagnostic compositions. comprising a. dry

mixture containing essentially the sodium bisulfite addition product of salicylaldehyde and a mem ber of the group consisting of the alkali metal-' and alkali-earth metal oxides and hydroxides, the proportions of the two materials-being such that the second member is present inexcess of the amount necessary to react with the first member in water to liberate salicylaldehyde and form a normal sulfite salt, and in excessto provide an alkaline solution containing said liberated salicylaldehyde upon additionof the composition to ,a liquid to be tested for a content of acetone and acetoacetic acid.

3. Diagnostic compositions comprising a dry sulfite addition product of salicylaldehyde and sodium hydroxide, the sodium hydroxide being present in quantity which is in excess of the amount necessary to react with the said addition product in water to liberate salicylaldehyde and acetic acid present in fluids which consists in adding to a measuredamount. of said fluid a measured amount of 'a composition as described -in claim Lwhereby, the resultant color oi the and acetotity of total acetone and acetoacetic acid in the fluid.

'6. A process for testing for acetone and acetoacetic acid present in body fluids which consists in adding to a predetermined amount of the body 5 of said alkali is in excess of the amount required to react with said addition product in water to liberate salicylaldehyde and form a normal sulfite salt, and in excess to provide an alkaline solution of said liberated salicylaldehydeupon addition of the said mixture to an aqueous fluid to be tested for acetone and acetoacetic acid, whereby to produce a color comparable with predetermined standard colors to indicate the percentage of ace- 10 tone and acetoacetic acid in the body fluid.

JONAS KAMLETQ 

